PCR In Situ Amplification and Catalyzed Signal Amplification. Approaches of Higher Sensitive, Non-Radioactive In Situ Hybridization.

Abstract

The method of in situ hybridization (ISH) using non-radioactive detection systems has become a very important molecular tool in research and diagnosis. However, nonradioactive ISH techniques generally detect only relatively abundant DNA or RNA molecules. The methods of target amplification, which multiply target molecules, or the methods of signal amplification, which increase the sensitivity of visualization, have been introduced for the detection of rare targets. Polymerase chain reaction (PCR) has been employed in most approaches to target amplification. Although the general principle of PCR in situ amplification is simple, its practical methods have revealed poor amplification efficiency and reproducibility as well as target localization. We have evaluated PCR in situ amplification methods to detect single copies of viral DNA with an experimental model of cervical carcinoma cell lines, and compared them with catalyzed signal amplification methods that we have developed using hapten-conjugated tyramide. Both amplification methods demonstrated the capability of detecting a single copy of DNA. In this overview, the applications and limitations of both target and signal amplification are summarized.